Cross-protection between antigenically distinct H1N1 swine influenza viruses from Europe and North America

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1 DOI: /j x Original Article Cross-protection between antigenically distinct H1N1 swine influenza viruses from Europe and North America Annebel R. De Vleeschauwer, a Sjouke G. Van Poucke, a Alexander I. Karasin, b Christopher W. Olsen, b Kristien Van Reeth a a Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. b Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, USA. Correspondence: Kristien Van Reeth, Laboratory of Virology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium. kristien.vanreeth@ugent.be Accepted 8 July Published Online 6 August Background An avian-like H1N1 swine influenza virus (SIV) is enzootic in swine populations of Western Europe. The virus is antigenically distinct from H1N1 SIVs in North America that have a classical swine virus-lineage H1 hemagglutinin, as does the pandemic (H1N1) 2009 virus. However, the significance of this antigenic difference for cross-protection among pigs remains unknown. Objectives We examined protection against infection with a North American triple reassortant H1N1 SIV [A swine Iowa H04YS2 04 (sw IA 04)] in pigs infected with a European avian-like SIV [A swine Belgium 1 98 (sw B 98)] 4 weeks earlier. We also examined the genetic relationships and serologic cross-reactivity between both SIVs and with a pandemic (H1N1) 2009 virus [A California (Calif 09)]. Results After intranasal inoculation with sw IA 04, all previously uninfected control pigs showed nasal virus excretion, high virus titers in the entire respiratory tract at 4 days post-challenge (DPCh) and macroscopic lung lesions. Most pigs previously infected with sw B 98 tested negative for sw IA 04 in nasal swabs and respiratory tissues, and none had lung lesions. At challenge, these pigs had low levels of cross-reactive virus neutralizing and neuraminidase inhibiting (NI) antibodies to sw IA 04, but no hemagglutination-inhibiting antibodies. They showed similar antibody profiles when tested against Calif 09, but NI antibody titers were higher against Calif 09 than sw IA 04, reflecting the higher genetic homology of the sw B 98 neuraminidase with Calif 09. Conclusions Our data indicate that immunity induced by infection with European avian-like H1N1 SIV affords protection for pigs against North American H1N1 SIVs with a classical H1, and they suggest cross-protection against the pandemic (H1N1) 2009 virus. Keywords Avian-like, cross-protection, H1N1 swine influenza virus, pandemic (H1N1) 2009, triple reassortant. Please cite this paper as: De Vleeschauwer et al. (2010) Cross-protection between antigenically distinct H1N1 swine influenza viruses from Europe and North America. Influenza and Other Respiratory Viruses DOI: /j x. Introduction Influenza viruses of H1N1, H3N2 and H1N2 subtypes are enzootic in swine populations worldwide, but they show different genetic and antigenic constellations in different parts of the world. 1 The complex epidemiology of swine influenza viruses (SIVs) is well illustrated by the nature of H1N1 SIVs in North America versus Europe. In North America, viruses of the classical swine H1N1 lineage were the predominant SIVs until These viruses are descendants of the first SIV isolated in 1930 and are related to the 1918 human pandemic H1N1 virus. Since 1998, reassortant H3N2 SIVs with genes of classical swine, avian and human influenza virus origin have become established in the swine population in North America. These viruses further reassorted with co-circulating classical H1N1 SIVs leading to the current triple reassortant H1N1 SIVs that contain a classical swine-lineage H1 hemagglutinin (HA). 2 In Europe, the prevailing H1N1 SIV is of entirely avian origin and therefore designated avian-like H1N1. It was introduced from wild ducks into the pig population in 1979 and has become the dominant H1N1 European SIV strain. 3,4 Surveillance studies show high seroprevalence rates for avian-like H1N1 SIVs in swine-dense regions of Western Europe. In Belgium, for example, 81% of sows and 42% of fattening pigs tested positive for antibodies. 5,6 In Asia, multiple H1N1 lineages appear to circulate, including avian-like, classical swine-lineage and triple reassortant H1N1 SIVs. 7,8 Reassortment between the avian-like and triple reassortant SIV lineages has been occasionally reported ª 2010 Blackwell Publishing Ltd, Influenza and Other Respiratory Viruses 1

2 De Vleeschauwer et al. in Thailand and China From May 2009 to May 2010, the pandemic (H1N1) 2009 influenza virus has been reported in swine in 22 countries in five continents. 11 This pandemic virus is as a reassortant of at least two circulating SIVs. Six gene segments, including the classical H1 HA, originate from North American triple reassortant SIVs, while the genes encoding the neuraminidase (NA) and matrix (M) proteins are closely related to those in European and or Asian avian-like SIVs. 12 The emergence of the pandemic (H1N1) 2009 influenza virus further complicates the SIV epidemiology. The HAs of avian-like and classical swine-lineage H1N1 SIVs are serologically distinct when compared by antigenic analyses with monoclonal antibodies. 13,14 Sequence alignment of the HA1 regions of the HA gene of these viruses likewise revealed several amino acid substitutions at putative antigenic sites. 14 The significance of these differences for cross-protection remains unknown, and there are no published cross-protection studies with H1N1 SIVs of different lineages in pigs. In this study, we aimed to examine a) to what extent immunity to a European avian-like H1N1 SIV may protect pigs from infection with a North American triple reassortant H1N1 SIV and b) the antigenic and genetic relationships between both viruses. In an attempt to extrapolate our findings to protection against the pandemic (H1N1) 2009 virus, a prototype pandemic virus was also included in the genetic and antigenic analyses. Materials and methods Viruses and sequence analysis A swine Belgium 1 98 (sw B 98) is representative of prevailing avian-like H1N1 SIVs in Western Europe (GenBank accession numbers ACN ). 15 A swine Iowa H04YS (sw IA 04) is a triple reassortant H1N1 SIV belonging to the North American H1ß (rh1n1-like) SIV cluster (GenBank accession numbers GQ ). 16 A California (Calif 09) is a prototype human pandemic (H1N1) 2009 virus (GenBank accession numbers ACP ). 12 The HA, NA, matrix (M1) and nucleoprotein (NP) of the 3 viruses were compared at the nucleotide and protein level by BLAST software (NCBI). Amino acid (aa) differences at putative antigenic sites of the HA, as defined by Brownlee and Fodor, 17 were identified by alignment (CLC Sequence Viewer 6) with the PR8 H1 reference strain. In total, 327 aa residues (H1 open reading frame numbering) were examined with special attention to 50 aa located at putative antigenic sites. Amino acid differences in the NA were identified by multiple alignment. In total, 469 NA aa residues were examined of which 195 were located in antigenic domains as described by Maurer-Stroh and co-workers. 18 Experimental design Twenty-five 6-week-old pigs from a conventional herd with a high health status and free of influenza A virus were used. The pigs were also serologically negative for porcine reproductive and respiratory syndrome virus and for porcine circovirus type 2. Pigs were randomly assigned to three groups. Two groups were inoculated initially with sw B 98 or sw IA 04 and challenged 4 weeks later with sw IA 04 (sw B 98-sw IA 04, n = 8 and sw IA 04-sw IA 04, n = 8). The third group served as a challenge control group and was challenged with sw IA 04 along with the previously sw B 98 or sw IA 04 inoculated pigs (sw IA 04-control, n = 9). All inoculations were performed intranasally at 7Æ0 log 10 egg infective doses 50% (EID 50 ). Inoculations were performed on unanesthetized animals. Pigs were held in a vertical position with the neck stretched. The inoculum was gradually instilled into the middle nasal cavity by insertion of a 15-mm plastic cannula attached to a syringe. All pigs were monitored daily for clinical signs from 5 days before until 7 days after initial inoculation (DPI), and from 5 days before until 7 days after challenge (DPCh), or until euthanasia. A daily clinical score was recorded for each pig as follows. A score of 1 each was given for the presence of fever (rectal temperature 40Æ0 C), depression, tachypnea (respiratory rate 45 per minute), dyspnea and forced abdominal respiration, resulting in a minimum clinical score of 0 and a maximum score of 5 per pig. To obtain the group clinical score per day, the individual scores of each day were added and divided by the maximum score possible (i.e. 40 for sw B 98- sw IA 04 and sw IA 04-sw IA 04, and 45 for sw IA 04- control). To determine the extent of virus excretion after the initial inoculations and after the challenge with sw IA 04, nasal swabs were collected daily from all pigs from 0 until 7 DPI, and from 0 until 7 DPCh, or until euthanasia. The swabs were weighed before and after collection to determine virus titers per 100 mg nasal secretions. To determine the extent of replication of the sw IA 04 challenge virus in the respiratory tract, 4 pigs per group were euthanized at 4 DPCh. Samples of the upper (nasal mucosa, tonsil and trachea) and lower (left and right lung) respiratory tract were collected for virus titration. Blood samples for serological examinations were collected at the start of the experiment and 4 weeks after the initial inoculation, i.e. at the time of challenge with sw IA 04. The remaining pigs were also bled at 14 and 28 DPCh. Virus titration Nasal swab samples from both nostrils were suspended in 1 ml of phosphate-buffered saline (PBS) supplemented with 10% fetal bovine serum, 100 IU ml penicillin and 100 lg ml streptomycin and mixed vigorously for 1 hour. 2 ª 2010 Blackwell Publishing Ltd, Influenza and Other Respiratory Viruses

3 Cross-protection between antigenically distinct H1N1 SIV The medium was collected, clarified by centrifugation and used for titration. Tissue samples were weighed and ground in PBS containing 10 IU ml penicillin and 10 lg ml streptomycin to obtain 10% or 20% (w v) tissue homogenates. The homogenates were clarified by centrifugation and used for titration. All samples were titrated on Madin Darby canine kidney (MDCK) cells in serum-free medium with trypsin (10 lg ml from porcine pancreas). Briefly, MDCK cells were seeded in 96-well cell culture plates at a concentration of cells per ml. After 3 days of incubation, the cells were 100% confluent and were inoculated with 10-fold dilutions of the samples using 4 wells per dilution. MDCK cells were observed daily for cytopathic effect until 7 days after inoculation. Virus titers were calculated by the method of Reed and Muench. 19 Serological assays Antibody titers against sw B 98, sw IA 04 and Calif 09 were determined in all sera by hemagglutination inhibition (HI), virus neutralization (VN) and neuraminidase inhibition (NI) assays, according to standard methods. 20,21 Before use, all sera were heat inactivated (56 C, 30 minutes). In the HI assay, sera were pre-treated with receptor-destroying enzyme from Vibrio cholerae and adsorbed onto chicken erythrocytes. Twofold serum dilutions were incubated [1 hour, room temperature (RT)] with four hemagglutinating units of the respective viruses. Finally, 0Æ5% chicken erythrocytes were added, and the assay was read after 1 h at RT. In the VN assay, twofold serum dilutions were incubated (1 hour, 37 C) with 100 TCID 50 of MDCK-adapted virus in microtiter plates. MDCK cells were added at a concentration of cells per ml. After incubation (24 hour, 37 C), the cells were fixed with 4% paraformaldehyde. Virus-positive cells were demonstrated by an immunoperoxidase staining using mouse monoclonal antibodies against influenza A virus nucleoprotein (HB65) and peroxidase-conjugated goat anti-mouse IgG. In the NI assay, standard virus doses were selected by an assay of NA activity based on the colorimetric analysis of sialic acid release from fetuin substrate. Twofold serum dilutions were incubated with the standard virus dilution in microtiter plates, and the reduction of NA activity in each serum dilution was compared with that in controls without serum. All sera were tested in duplicate, and antibody titers were expressed as the reciprocal of the highest serum dilution that completely inhibited hemagglutination or virus replication in MDCK cells, or that gave a 50% inhibition of NA activity. Starting dilutions were 1:2 in the VN test, and 1:10 in the HI and NI tests. Statistical analysis Differences in serum HI, VN and NI antibody titers were compared between groups in two-sample Student s t-tests. Samples that tested negative in the serological assays were given a value corresponding to half of the minimum detectable antibody titer. P <0Æ05 was taken as the level of statistical significance. Results Genetic and antigenic relationships between sw B 98, sw IA 04 and Calif 09 Percentages of nucleotide and aa identity between the 4 analyzed genes of sw B 98 and sw IA 04 or Calif 09 are shown in Table 1. Nucleotide sequence identity of the HA gene of sw B 98 and the 2 viruses with the classical swinelineage HA (sw IA 04 and Calif 09) was low (74 75%), whereas both classical HAs were more similar (91%). Nucleotide sequences of the NA and M1 genes of sw B 98 were more similar to Calif 09 (92% and 95%, respectively) than to sw IA 04 (79% and 88%, respectively), consistent with the Eurasian virus phylogenetic lineage of these 2 genes in the 2009 pandemic viruses. All viruses were equally similar in the NP gene (83%). Similar but generally higher relationships were observed at the aa level. Amino acid changes at presumed antigenic sites of the HA are shown in Figure 1. The HA1 segment of sw B 98 contained 76 aa differences compared to sw IA 04 and 84 aa differences with Calif 09, and respectively 13 and 14 changes were in aa residues at putative antigenic sites. The HA1 regions of the viruses with the classical swine-lineage HA genes were more closely related (39 aa differences, with 8 at putative antigenic sites). The NA gene of sw B 98 was more closely related to Calif 09 (28 aa differences, with 12 at putative antigenic sites) than to sw IA 04 (82 aa differences, with 27 at putative antigenic sites). The NA of sw IA 04 and Calif 09 was different in 83 aa residues, of which 31 were located at putative antigenic sites. Table 1. Percent identity of the nucleotide and amino acid sequences of the hemagglutinin (HA), neuraminidase (NA), matrix (M) and nucleoprotein (NP) genes of sw B 98 with those of sw IA 04 or a prototype pandemic (H1N1) 2009 virus % identity compared to sw B 98 HA NA M1 NP N aa N aa N aa N aa Sw IA Calif N, nucleotide; aa, amino acid. ª 2010 Blackwell Publishing Ltd, Influenza and Other Respiratory Viruses 3

4 De Vleeschauwer et al. Figure 1. Alignment of deduced amino acid sequences at antigenic sites, as defined by Brownlee and Fodor, 17 of the hemagglutinins of sw B 98, sw IA 04 and Calif 09. Only the amino acids different from those in the sw B 98 sequence are indicated, and conserved residues are shown as dashes. Virus excretion and serological response after initial infection with sw B 98 or sw IA 04 Mild fever (40Æ4 40Æ8 C) was seen in all pigs 1 and 2 days after inoculation with sw B 98 (clinical scores 0Æ30 and 0Æ20 on 1 and 2 DPI, respectively), and in most pigs after initial inoculation with sw IA 04 (clinical scores 0Æ20 and 0Æ10 on 1 and 2 DPI, respectively), but respiratory signs were absent. All inoculated pigs excreted the SIVs used for inoculation for 6 consecutive DPI. Mean virus titers in nasal swabs are shown in Figure 2. The challenge control pigs remained virus negative. All pigs were negative in antibody assays at the start of the experiments against all three viruses. Serological findings at the time of challenge with sw IA 04, i.e. 4 weeks after initial inoculation, are shown in Table 2. As expected, all control pigs were seronegative at the time of challenge, whereas all pigs inoculated previously with either sw IA 04 or sw B 98 had HI, VN and NI antibodies to the SIV used for inoculation. In addition, most pigs inoculated with sw IA 04 had HI antibodies that cross-reacted with sw B 98 and or Calif 09, and all these pigs had cross-reactive VN and NI antibodies to the other two viruses. Cross- Figure 2. Nasal virus excretion after initial inoculation and after challenge with sw IA 04. Mean virus titers in nasal swabs of each group are given. Sw B 98-sw IA 04, sw IA 04-sw IA 04 and sw IA 04-control groups are represented by dotted, dashed and full lines, respectively. The thin dashed line represents the detection limit (<1Æ7 log 10 TCID mg). 4 ª 2010 Blackwell Publishing Ltd, Influenza and Other Respiratory Viruses

5 Cross-protection between antigenically distinct H1N1 SIV Table 2. Serological findings at the time of challenge with sw IA 04 and 2 weeks later in naïve control pigs and sw B 98 or sw IA 04-immune pigs Mean antibody titers of positive pigs ± SEM* (number of positive pigs total number of pigs) Time of challenge 14 DPCh Group Test sw B 98 sw IA 04 Calif 09 sw B 98 sw IA 04 Calif 09 sw IA 04- control HI < < < 18 ± 2 (5 5) 68 ± 12 (5 5) 10 ± 0 (2 5) VN < < < 46 ± 15 (5 5) 154 ± 33 (5 5) 12 ± 3 (5 5) NI < < < 22 ± 7 (5 5) 288 ± 93 (5 5) 16 ± 6 (5 5) sw IA 04-sw IA 04 HI 16 ± 2 (7 8) 88 ± 22 (8 8) 15 ± 2 (4 8) 15 ± 3 (4 4) 45 ± 13 (4 4) 15 ± 4 (2 4) VN 44 ± 13 (8 8) 180 ± 26 (8 8) 9 ± 2 (8 8) 75 ± 40 (4 4) 208 ± 31 (4 4) 27 ± 14 (4 4) NI 25 ± 6 (8 8) 355 ± 91 (8 8) 16 ± 4 (8 8) 30 ± 6 (4 4) 280 ± 40 (4 4) 23 ± 6 (4 4) sw B 98-sw IA 04 HI 135 ± 31 (8 8) < < 120 ± 23 (4 4) 40 ± 14 (4 4) 17 ± 3 (3 4) VN 416 ± 82 (8 8) 8 ± 2 (7 8) 3 ± 1 (7 8) 1216 ± 352 (4 4) 128 ± 23 (4 4) 32 ± 6 (4 4) NI 230 ± 70 (8 8) 29 ± 4 (8 8) 68 ± 15 (8 8) 800 ± 160 (4 4) 400 ± 139 (4 4) 720 ± 201 (4 4) *Standard error of the mean; antibody titers below detection limits, i.e. <10 in HI and NI tests and <2 in VN test. HI, hemagglutination inhibition; NI, neuraminidase inhibition; VN, virus neutralization. reactive HI, VN and NI antibody titers to both heterologous viruses were significantly lower than those to the homologous virus (P <0Æ05). All pigs inoculated with sw B 98 lacked cross-reactive HI antibodies to sw IA 04 and Calif 09, but most of them had cross-reactive VN antibodies to both viruses, though at lower titers than to the homologous virus (P < 0Æ05). Cross-reactive NI antibodies against both viruses were found in all pigs, and they were higher to Calif 09 than to sw IA 04 (P <0Æ05). Clinical and virological protection after challenge with sw IA 04 After subsequent challenge with sw IA 04, mild fever (40Æ6 40Æ1 C), depression and respiratory signs were seen in the previously uninfected control pigs at 1 (clinical score 0Æ44) and 2 (clinical score 0Æ17) DPCh only. Mean virus titers in nasal swabs are shown in Figure 2. All previously uninfected control pigs excreted the sw IA 04 challenge virus at high titers (up to 7Æ5 log 10 TCID mg) for 6 consecutive DPCh or until euthanasia at 4 DPCh, whereas disease and virus excretion were undetectable in the sw IA 04-sw IA 04 group. Interestingly, only 1 of 8 pigs inoculated previously with sw B 98 had mild fever (40Æ0 40Æ1 C) at 1 and 2 DPCh with sw IA 04 (clinical score 0Æ03 on both days). Likewise, only 2 of 8 pigs of the sw B 98-sw IA 04 group had detectable nasal virus excretion, and nasal virus titers were lower and detectable for a shorter duration than in the previously uninfected control group. One pig tested positive at 3 DPCh (3Æ0 log 10 TCID mg) only, and one pig (nr V ) on 2, 3 and 4 DPCh (1Æ7, 3Æ0 and 2Æ3 log 10 TCID mg, respectively). Sw IA 04 virus titers in the respiratory tract of the pigs euthanized at 4 DPCh are shown in Table 3. Sw IA 04 was recovered from the nasal mucosa, tonsil, trachea, left lung and right lung of all 4 previously uninfected control pigs. Table 3. Virus titers in the respiratory tract 4 days after challenge with sw IA 04 in naïve control pigs and sw B 98 or sw IA 04-immune pigs Range of virus titers (log 10 TCID 50 g) of positive pigs (number of positive pigs total number of pigs) Group Nasal mucosa respiratory part Nasal mucosa olfactory part Tonsil Trachea Left lung Right lung sw IA 04-control 4Æ1 6Æ6 (4 4) 4Æ3 6Æ0 (4 4) 4Æ0 5Æ0 (4 4) 5Æ5 6Æ5 (4 4) 6Æ3 6Æ7 (4 4) 6Æ2 7Æ2 (4 4) sw IA 04-sw IA 04 <* < < < < < sw B 98-sw IA 04 < < < 2Æ0 (1 4) < < *Virus titers below the detection limit (1Æ9 log 10 TCID 50 g for nasal mucosa, tonsil and trachea; 1Æ7 log 10 TCID 50 g for lung) in all pigs. ª 2010 Blackwell Publishing Ltd, Influenza and Other Respiratory Viruses 5

6 De Vleeschauwer et al. Virus titers ranged from 4Æ0 to 6Æ6 log 10 TCID 50 g in the upper respiratory tract, and from 6Æ3 to 7Æ2 log 10 TCID 50 g in the lungs. All four pigs of the sw IA 04-sw IA 04 group were completely virus negative. In the sw B 98-sw IA 04 group, only pig nr V tested positive, and this was only in the tracheal sample and only at a low titer of 2Æ0 log 10 TCID 50 g. Areas of lung consolidation, involving 5 38% of the lung surface, were present in the four previously uninfected control pigs euthanized, but absent in the pigs from the other groups. Serological profile after challenge with sw IA 04 Serological findings at 14 DPCh are shown in Table 2. All previously uninfected control pigs seroconverted (i.e. 4- fold increase in antibody titer) to sw IA 04 after challenge infection and developed cross-reactive HI, VN and NI antibody titers to both other viruses, though at lower titers than the homologous sw IA 04 titers. Only two pigs, however, developed HI antibodies to Calif 09. In the sw IA 04-sw IA 04 group, HI, VN and NI antibody titers to the three viruses were comparable to those before challenge (P >0Æ05). In the sw B 98-sw IA 04 pigs, antibody titers to sw B 98 did not change after challenge (P >0Æ05). Antibody titers to sw IA 04 and sw Calif 09 increased, but only the increases in VN antibody titers to sw IA 04, and VN and NI antibody titers to Calif 09 were significant (P <0Æ05). Antibody titers at 28 DPCh were similar to those at 14 DPCh (P >0Æ05). Discussion This study shows a stronger than expected protection against infection with a North American triple reassortant H1N1 SIV in pigs infected with a phylogenetically distinct European avian-like SIV 4 weeks earlier. It has long been known that the HA1 of both virus lineages shows considerable antigenic and genetic differences. 13,14,22 Cross-reactive HI antibodies against viruses with a classical H1 are usually only detectable in high titered or hyperimmune antisera against avian-like H1N1 SIV, and not in lower titered postinfection swine sera. 3,16,23 The VN test detects a broader range of neutralizing antibodies than the HI test, 24 which likely explains the cross-reactive VN antibody titers in our study. Dürrwald et al. 25 detected cross-reactive VN antibodies against German isolates of the pandemic 2009 (H1N1) virus in pigs experimentally infected with the avian-like H1N1 SIV A swine Haselünne IDT , which is in agreement with our findings. The cross-protection between European and North American H1N1 SIVs was clearly stronger than that observed between European H1N1 and H1N2 SIVs in previous experimental studies. 20,26,27 In those prior studies, the pigs were first inoculated with sw B 98 followed 4 weeks later by sw Gent , a typical European H1N2 virus. One of these studies used the same intranasal inoculation route and dose as in the present experiment. 27 This resulted in high H1N2 virus titers in nasal swabs of all challenge control pigs during 6 consecutive DPCh. All pigs that had been previously infected with sw B 98 also shed high amounts of H1N2 virus in nasal swabs, and the duration of excretion was only 1 or 2 days shorter than in the control group. This contrasts with the excretion of the North American H1N1 SIV in the present study, which was largely blocked in sw B 98-immune pigs. The inferior protection against the H1N2 virus may relate to its more distant relationship to both the NA and HA of sw B 98 when compared with the North American H1N1 SIV. Indeed, the HA of sw Gent (H1N2) shows only 70Æ5% sequence homology with sw B 98 (H1N1) and as much as 28 aa differences in antigenic sites, versus 75% homology and 13 aa changes in antigenic sites for sw IA 04 (H1N1). This translates into detectable cross-reactive VN and NI antibodies to the antigenically more closely related H1N1 virus, but not to H1N2. 20,27 The NP and M proteins, which are major targets for T cells, show more than 95% nucleotide identity between European H1N1 and H1N2 SIVs, 26 but they remain relatively conserved in the North American H1N1 SIV. Based on these findings and on general knowledge of the cross-protective immune response between influenza viruses, 28 we believe that the observed cross-protection between antigenically distinct H1N1 SIVs in the present study results from a combination of antibody- and cell-mediated immune (CMI) responses to multiple viral proteins. In addition, mucosal as well as systemic immunity is likely involved. Most important, our data provide further proof of the concept that cross-protection can occur between influenza viruses with multiple aa differences in three of four antigenic sites of the HA, and in the absence of detectable cross-reactive serum HI antibody. Our data further support the concept advanced previously 16 that the immune response after infection with avianlike European H1N1 SIV may also protect pigs against the pandemic (H1N1) 2009 virus. Our genetic data and serologic results agree with previous findings about the swine ancestry of the pandemic virus. 10,12 The HA and NP of the pandemic Calif 09 isolate were closely related to the North American sw IA 04 SIV, and both viruses shared many aa changes at antigenic sites of the HA, compared to the European sw B 98 SIV. The NA and M1 of the pandemic virus, in contrast, were phylogenetically derived from Eurasian-lineage, avian-like SIV. 12 This was reflected in higher NI antibody titers to the pandemic virus than to the North American challenge H1N1 SIV in pigs immune to sw B 98. It is therefore rational to expect an even more solid cross-protection against the pandemic virus than to sw IA 04-like North American swine viruses in response to prior infection with 6 ª 2010 Blackwell Publishing Ltd, Influenza and Other Respiratory Viruses

7 Cross-protection between antigenically distinct H1N1 SIV sw B 98-like European viruses, though this assumption needs to be tested by further challenge studies. In experimental studies, in influenza naive pigs, the pandemic virus was as infectious for pigs as the endemic SIVs, and it was readily transmitted between pigs At this time of writing, however, cases of pandemic (H1N1) 2009 virus in pigs in Europe have been reported in only 10 countries, and the virus is far less widespread than the endemic SIVs. 11 However, it is striking that most cases have been reported in countries that were previously free of SIV, such as Norway, 33 and in countries with a low-to-moderate SIV prevalence, like Ireland and the United Kingdom. The extent of cross-protection between any two influenza viruses may appear greater under experimental than under natural conditions, because of many possible differences between the experimental and field situations. As an example, the short time interval between the primary influenza virus inoculation and challenge in our and other studies represents an artificial situation and may optimize the outcome of the experiment. On the other hand, fattening pigs in swine-dense regions of Europe are frequently exposed to multiple SIV subtypes within their 26-week short lifetime. For instance, as many as 84Æ5% of fattening swine farms in Belgium, France, Italy and Spain showed serologic evidence of infection with 2 or 3 SIV subtypes during Such consecutive or co-infections will likely increase crossprotection against viruses with a classical H1 HA. Experimental consecutive infections with European H1N1 and H1N2 viruses were also shown to induce cross-reactive HI antibody to North American SIVs as well as pandemic (H1N1) 2009 virus, whereas the respective single infections failed to do so. 16 Such combined H1N1-H1N2 infections even afforded protection against challenge with H3N2 SIV, despite no or minimal cross-reactive HI antibody. 27 Recent serological investigations demonstrated cross-reactive HI antibodies to the pandemic 2009 (H1N1) virus in as much as 52% of 1559 pig serum samples from 195 German pig herds. 25 The sera had been collected in the mid-2009, before the first reports of the pandemic virus in European swine populations. Furthermore, vaccines based on the endemic European SIVs are licensed in the main pig-producing Member States of Europe, but not in countries with lower pig numbers and SIV prevalences like Norway or Ireland. These vaccines seem to offer partial cross-protection against the pandemic virus. 25 All these data further support the idea that pigs in swine-dense regions of Europe may experience protection against influenza viruses with a classical swine-lineage H1. Acknowledgements We thank Ruben Donis (Centers for Disease Control and Prevention) for providing the Calif 09 virus, and Lieve Sys, Melanie Bauwens, and Philippe Elskens for excellent technical assistance. This study was supported by the 6th Framework Research Program of the European Commission (FLUINNATE SSPE-CT ). References 1 Olsen CW, Brown IH, Easterday BC, Van Reeth K. Swine influenza; in Straw B, D Allaire S, Zimmerman J, Taylor D (eds): Diseases of Swine. Iowa: Iowa State University Press, 2006; Olsen CW. The emergence of novel swine influenza viruses in North America. Virus Res 2002; 85: Pensaert M, Ottis K, Vandeputte J, Kaplan MM, Bachmann PA. 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